CD63 is a member of the transmembrane-4 glycoprotein superfamily, known as tetraspanins. The tetraspanins contain four transmembrane domains, two extracellular loops flanked by short intracellular N and C termini, and a highly conserved sequence motif within the larger extracellular domain. Tetraspanins interact among themselves and with other transmembrane proteins to form membrane domains denoted tetraspanin-enriched microdomains (TEMs). At least in part through the TEMs, tetraspanins regulate a variety of cellular processes including cell fusion, intracellular trafficking, cell adhesion, motility, invasion, and cell differentiation. CD63 is among the most highly expressed tetraspanins in endothelial cells (ECs). Still, the role of CD63 in endothelial biology and angiogenesis remains to be addressed. We show that CD63 is co-localized with the type I integral lysosomal membrane protein (LAMP-1) but also present on the EC surface. By silencing CD63 expression in primary ECs, we demonstrate that CD63 associated with both β1 integrin and VEGFR2 in the plasma membrane and was required for VEGFR2-β1 integrin complex formation. Loss of CD63 expression disrupted downstream signaling path ways both in endothelial cells in culture and in intact tissues. As a consequence, CD63-deficient ECs failed to engage in sprouting angiogenesis and organize into tube structures.